Designer Liposomic Nanocarriers Are Effective Biofilm Eradicators

Drug delivery via nanovehicles is successfully employed in several clinical settings, yet bacterial infections, forming microbial communities in the form of biofilms, present a strong challenge to therapeutic treatment due to resistance to conventional antimicrobial therapies. Liposomes can provide a versatile drug-vector strategy for biofilm treatment, but are limited by the need to balance colloidal stability with biofilm penetration. We have discovered a liposomic functionalization strategy, using membrane-embedded moieties of poly[2-(methacryloyloxy)ethyl phosphorylcholine], pMPC, that overcomes this limitation. Such pMPCylation results in liposomic stability equivalent to current functionalization strategies (mostly PEGylation, the present gold-standard), but with strikingly improved cellular uptake and cargo conveyance. Fluorimetry, cryo-electron, and fluorescence microscopies reveal a far-enhanced antibiotic delivery to model Pseudomonas aeruginosa biofilms by pMPC-liposomes, followed by faster cytosolic cargo release, resulting in significantly greater biofilm eradication than either PEGylation or free drug. Moreover, this combination of techniques uncovers the molecular mechanism underlying the enhanced interaction with bacteria, indicating it arises from bridging by divalent ions of the zwitterionic groups on the pMPC moieties to the negatively charged lipopolysaccharide chains emanating from the bacterial membranes. Our results point to pMPCylation as a transformative strategy for liposomal functionalization, leading to next-generation delivery systems for biofilm treatment.


P. 23
References P. 24 The potential toxic effect of the different liposomal formulations tested was expressed as a viability percentage calculated using the following formula:

Evaluation of cytotoxicity
Where ODtest was the optical density of those wells treated with the liposomes solutions, and ODc was the optical density of those wells treated with supplement-free DMEM media.

Pyocyanin quantitation assay
Pseudomonas aeruginosa (PA) biofilms were grown on 24-well plate. 1 mL of the inoculum was grown in a humidified incubator for 24 h at 37 ˚C without shaking. After 24h, the fully formed biofilm was gently washed two times with PBS. 1 mL of liposomal suspension or BM2G medium was added to each well and subsequently the samples were incubated at 37 ˚C for 4 h.
Each well was then washed with PBS and second does (1 mL) of antibacterial treatment was applied for 4 h with incubation at 37 ˚C. Subsequently 24-well plate was sonicated at room temperature for 5 min. The collected bacteria suspension were centrifuged at 4500 rpm for 20 min. Obtained supernate was filtrated through 0.22 µm syringe filter. 0.5 mL of supernate was mixed with 0.17 mL of chloroform and shaken vigorously. Subsequently, samples were centrifuged at 4500 rmp for 10 min. The organic phase was transfer to new tubes and mixed with 0.12 mL of 0.2 M HCl and shaken vigoursly. The Samples were again centrifuged at 4500 rpm after which water phase was collected and pyocyanin was quantified using HPLC analysis.

Pyocyanin analysis by HPLC
Samples and pyocyanin standard solutions were prepared in 0.2 M HCl, supplemented with 1,5-Naphthalenediamine (0.1 mg/mL) as internal standard and filtered through 0.2 mm PTFE filters (StarTech®, CA) prior to HPLC analysis. Sample solutions were kept at 4°C prior to injection and were separated by reverse-phase chromatography on a Gemini C18 (4.6 x 150 mm, 5 mm) column (Phenomenex©, USA). Chromatography was performed on a Prominence UFLC LC-20AD system (Shimadzu©, Japan) consisting of a SIL-20AC autosampler (Shimadzu©, Japan), CTO-20AC column oven (Shimadzu©, Japan) and a SPD-M20A diode array detector (Shimadzu©, Japan). Elution was done using an isocratic gradient of 70% solvent A (0.04% TFA in water) and 30% solvent B (10% water and 0.04% TFA in acetonitrile) at a flow rate of 1 mL per minute for 5 minutes at 25 ˚C, while monitoring at 388nm. Data analysis was performed using LabSolutions ver. 5.97 (Shimadzu©, Japan).

Stability of loaded liposomes
Liposomes with encapsulated drug were store at 4 ˚C, and at define time points, liposomal suspension was dialyzed (dialysis bag with 50 kDa cutoff) against Na 2 SO 4 (pH 7.0, ~320 mOsmo/kg). Amount of remining drug encapsulated in liposomes was determined using ultraviolet spectrophotometer (Cary 100 Bio, Varian Inc, USA).